Fixation Standard

8/6/2019 Fixation Standard

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Standard fixation and embedding protocol for resin section TEM

Bio-Imaging, SWDSOP, 2004

The main purpose of fixation is:1) to cross-link cellular structures into a matrix so as to preserve the structure of the cells withthe minimum of alteration from the living state (i.e. with no changes in morphology, volume, orspatial relationships, and with minimum loss of cellular constituents) and,2) to protect and stabilize cellular structures from changes during subsequent treatments andfrom irradiation by the electron beam.

Fixation is the first and most important step in any EM study, since mistakes made at thispoint render the whole project useless. Below are a number of fixation protocols that shouldprovide good starting points for most EM studies.

SAFTEY NOTE: Most of the chemicals used for processing specimens for electronmicroscopy are extremely hazardous

Glutaraldehyde, formaldehyde and osmium tetroxide are volatile and can fix anycells/tissues they contact (they will fix your respiratory epithelium, corneas, and epithelial cellson your hand, etc): it is therefore essential to use them in a fume hood. Although dangerousthese fixatives cannot penetrate more than 1-2 mm into tissues, so exposure to them rarelycauses any permanent damage.

The epoxy resins used to embed the specimens are potentially more dangerous thanthe fixatives. Many of the resin components are known to cause cancer in rats or mice. Duringthe embedding process the resins are dissolved in solvent(s) that can carry the resin into your

skin EVEN through plastic gloves. In contrast to fixatives, whose actions are immediate andapparent, the consequences of exposure to the resins are not apparent for years. Thereforebe careful with all resins prior to polymerization into hard blocks.

A) Preparation schedule for tissues cut into 1-2 mm thick sheets or


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Resin embedding (Epon mix)Propylene oxide 2 changes, each of 15 minutes(N.B. since propylene oxide is much more volatile than ethanol or acetone be careful not toallow the sample to be exposed to the air as damage will occur due to the rapid evaporationof the solvent)

Resin infiltration 2:1 mix of propylene oxide:resin 1 h

1:1 mix of propylene oxide:resin 1 h1:2 mix of propylene oxide:resin 1 h100% resin overnight

Change in to fresh resin 1 h

Embedding Fresh resin in embedding moulds remember to label the samples.Polymerise 12-24 hours at 60-70C

The above protocol uses phosphate buffer (not PBS) as the buffering vehicle for theglutaraldehyde and osmium fixatives. As alternatives cacodylate, PIPES or HEPES buffer canbe used instead.The type of buffer in which the fixatives are made up can affect the appearance of thespecimen. However, one advantage of cacodylate and PIPES or HEPES is that CaCl2 and/or

MgCl2 can be added to the primary fixative. Calcium (and Mg) ions reduce the extraction ofcellular components and also enhance the retention of phospholipids.

B) Fixation protocol for cells in suspension

There are a number of ways to fix cells grown in suspension.i) gently pellet the cells (800 g for 5-10 min), remove most of the culture medium and then fixthe cells by adding an excess volume (5-10 times the cell volume) of fixative and re-suspendthe cells in the fixative. After 10 min the cells should be pelleted in an eppendorf tube (3-5min at max rpm), the fixative removed and replaced by fresh fixative.ii) cells can be fixed by adding 1 volume of 25% glutaraldehyde to 9 volumes of cellsuspension in the original culture medium. After 5-10 min the cells can be pelleted andfixation continued in the original fixative. Alternatively, after pelleting, the original fixative canbe replaced by a standard glutaraldehyde fixative and the fixation process continued for therequired time.

After the initial fixation step the sample(s) can be further processed as described above. Thecells do not need to be re-suspended during the subsequent steps and if treated carefully thepellet can be freed from the tube and flat embedded allowing sections to be cut throughwhich ever part of the pellet is required.

C) Fixation protocol for cells grown in culture dishes

Primary fixationAfter removal of most of the culture medium, the cells should be fixed with an aldehyde-basedfixative as described in protocol A.

After the initial fixation the cells can be further processed by a number of different waysdepending on the aim(s) of the experiment.

i) the cells can be released from the plastic by gentle scraping and then pelleted. Thesamples can then be post-fixed and processed as normal.

ii) the samples can be post-fixed with osmium tetroxide (and uranyl acetate if required) andthen dehydrate through a series of ethanols and embedded in situ.N.B. In this case, since most plastics dissolve in acetone and propylene oxide the samplesmust be dehydrate using ethanol and a series of resin-ethanol mixes used during theinfiltration process (instead of the more usual resin-propylene oxide mixes).


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iii) the samples can be further processed and dehydrated using ethanol as described above.However, after fully dehydrating the samples, the cells can be released from the plastic usingpropylene oxide. By gentle pipetting propylene oxide over the cells they will detach from theplastic either as individual cells or in sheets. The detached cells should be pelleted, washedseveral times with propylene oxide to remove and solubilized plastic, and then embedded asdescribed above.

D) Fixation protocol for pathology samples

Fix tissues in a mixture of 2.5% glutaraldehyde, 2% (para)formaldehyde in 100 mMcacodylate buffer (pH 7.0) with 2 mM CaCl2.After an initial ~30 min fixation, cut the specimens into small (~1 mm

3) pieces and continue

fixation in fresh fixative for 16-24 h at 4C.Wash briefly with 200 mM cacodylate buffer (pH 7.0).

Post-fix with 1% osmium tetroxide in 100 mM cacodylate buffer (pH 7.0); 2 h at 4C.Wash with excess distilled water [to remove any free cacodylate and/or phosphate ions].

En bloc stain with 2.0% aqueous uranyl acetate, ~2 h at 4C (in dark).Dehydrate with acetone (or ethanol), propylene oxide and embed in resin as outlined above.

General references for TEM

Glauert, A.M., Lewis, P.R. (1998). Biological Specimen Preparation for TransmissionElectron Microscopy. Practical Methods in Electron Microscopy: vol. 17. This tells you allabout the fixatives, buffers etc. and also covers dehydration and embedding.

Reid, N., Beesley, J.E. (1991). Sectioning and Cryosectioning for Electron Microscopy.Practical Methods in Electron Microscopy: vol. 13.This book covers section cutting for both conventional resin sections as well as cryosections,although the Griffiths book (see below) is the best for cryosectioning and cryo-immunocytochemistry.

Lewis, P.R., Knight, D.P. (1977). Staining Methods for Sectioned Material. Practical Methodsin Electron Microscopy: vol. 5.This is a useful reference for section staining etc. You can get the actual references for thelead citrate and uranyl acetate staining protocols from here if you want to quote the originalpapers.

Griffiths, G. (1993). Fine Structure Immunocytochemistry. Springer-Verlag.Although this book is mainly about immunocytochemistry (and especially the techniques ofcryosectioning) there are useful chapters on the chemistry of aldehyde fixatives.

All EM supplies are from Agar Scientific or TAAB Laboratories these are 2 of the mainsuppliers of EM materials in the UK.

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Fixation Standard